This is a revised Stage II CEBRA application, a competing renewal and extension of our Stage I program to develop a new model for HIV-1 infection in the CNS employing immunocompetent, conventional mice. In Stage I we demonstrated for the first time that there HIV-1 can replicate in several types of primary murine cells and we constructed EcoHIV, a chimeric HIV-1 capable of entry, replication, and spread in mouse lymphocytes. Importantly, since the first submission, we found that mice inoculated i.v. with EcoHIV become persistently infected at high frequency as indicated by seroconversion and presence of virus in the spleen and peritoneal macrophages over a 3 month period. Some animals showed evidence of virus infiltration into the brain, elevated expression of genes associated with inflammation in brain tissue, and brain lesions one of which contained structures that resembled multinucleated giant cells seen other retroviral neuropathies. We believe that these results bring us closer to the proof-of-principle of the proposed model. We have revised the application in light of these findings and the Reviewers' suggestions with an overall goal of investigating HIV-1 infection and pathogenesis of the nervous system in mice. We plan to identify the similarities between HIV-1 neuropathogenesis in this model and human disease and to employ them to investigate in detail the roles of HIV-1 replication dynamics and viral envelope in initiation and progression of brain disease. Initial studies on the effect of morphine in this model are also proposed. The Specific Aims are: 1) To characterize systemic EcoHIV infection and virus entry into the brain in immunocompetent inbred mice; 2) To establish an experimental paradigm of HAD-like neuropathogenesis in the EcoHIV mouse model; 3) To investigate contribution of specific HIV-1 gp120 domains to neuropathogenesis in the EcoHIV mouse model; 4) To investigate HIV-1 mediated neuropathogenesis in the mouse as a function of EcoHIV-1 virulence; 5) (Exploratory Aim): To explore modulation of HIV-1 replication in mice by morphine. We shall investigate EcoHIV replication in vitro and in vivo using DNA- and RNA PCR, Elisa, and immunohistochemistry coupled with laser capture microdissection. Pathological changes will be scored by microscopy of tissue sections, by flow cytometry, and by evaluation of changes in protein and gene expression in the brain by IHC, Elisa, RNA- and real time-PCR, and protein arrays. This Program will develop and test chimeric HIV-1 species competent to replicate and induce disease in any conventional mouse and meets the CEBRA criteria as high impact/high risk research. This model will be valuable for studies of HIV-1 neuropathogenesis, impact of addiction and drugs of abuse, as well as development of therapies and vaccines.